Project Summary Inflammation serves a protective role in the body to fight infection and mediate wound healing; however, the inflammatory response can become deleterious if left uncontrolled or unresolved. Most pathology, including atherosclerosis, vasculitis, and ischemia/reperfusion injury?which can exacerbate potential comorbidities such as myocardial infarcts and stroke?is due to dysfunctional inflammation. Targeting inflammation could thus provide the means to combat inflammatory disease. This objective can be realized by targeting a common process that underlies all inflammation: the directed movement of white blood cells (WBCs) from circulation into affected tissue. In this process, transendothelial migration (TEM), WBCs migrate between lateral borders of endothelial cells (ECs) lining blood vessels to enter sites of inflammation. TEM is a critical step in the inflammatory response as it is the ultimate step preceding WBC access to inflamed tissue. Successful blocking of TEM would preclude tissue damage in an inflammatory context and is thus a therapeutic goal. TEM is mediated by interactions between surface adhesion molecules on the WBC and ECs, resulting in signaling within ECs that traffics membrane from the EC lateral border recycling compartment (LBRC), a perivascular vesiculotubular structure, to the site of TEM. This provides additional surface area required for WBC migration between adjacent ECs. Trafficking of LBRC is the final common pathway for TEM regardless of WBC type or inflammatory stimulus. Blocking critical adhesion molecules or downstream EC signaling pathways blocks TEM of WBC by 80-90% in vitro and in vivo. However, residual transmigration rate remains 10-20%, raising the question as to whether specific subpopulation(s) of WBC are resistant to TEM blockade and whether non-blocked WBCs use alternative signaling pathways to recruit the LBRC to transmigrate. AIM I will phenotypically characterize monocytes that overcome TEM blockade to determine if they represent a distinct monocyte subset. I will interrogate the mechanism by which these cells transmigrate in an in vitro TEM model and in murine models of inflammation. This will allow for the identification of surface receptors/signaling pathways that can be targeted to block the exit of all circulating monocytes from the vasculature in situations in which blocking 100% of inflammation is critical. AIM II will interrogate mechanisms that allow these monocytes to use alternative transmigration pathways. I will utilize a guided in vitro screening approach and in vivo real time intravital confocal microscopy to validate my in vitro results by inspecting mechanisms functioning at the TEM step directly. The project will shed light on alternative TEM pathways that may be important under some pathologic conditions and alert us to ways in which the body may compensate for the chronic blockade of traditional TEM pathways.